DNA typing to identify native inland Oncorhynchus mykiss
A. Abstract and Statement of Innovation
Native inland populations of rainbow trout, Oncorhynchus mykiss , particularly the resident populations of the species, have often been found to have hybridized with introduced populations of the widely-cultured coastal form of the species. These groups have historically been differentiated using allozyme typing, which has limitations both with respect to ease of sampling and power of discrimination.
We propose to detect DNA polymorphisms differentiating inland and coastal populations using AFLP
(amplified fragment length polymorphism) analysis of both genomic DNA and cDNA samples. This approach, which has been used successfully in our laboratory for identify sex-associated DNA markers, should result in simple, efficient methods which can be used in a large number of laboratories to identify levels of hybridization within inland rainbow trout populations. The project is innovative in that it uses novel approaches to develop new DNA tests that should be useful in management programs in the region.
Gary Thorgaard will serve as principal investigator, providing overall project supervision and coordination, and Joseph Brunelli, assistant research professor, will be co-principal investigator and will insure that the molecular biology methods are applied successfully.
B. Technical and Scientific Background
Native inland populations of rainbow trout, Oncorhynchus mykiss , particularly the resident populations of the species, have often hybridized with introduced populations of the widely-cultured coastal form of the species. This reflects historic practices of widespread introduction of the coastal form of the species, which has been widely cultured in hatcheries (Behnke, 1992). Present practices are more conservative and in some cases include limiting introductions of hatchery fish to sterile individuals
(Dillon et al., 2000). In addition to greater ability of the wild fish to thrive in the natural environment, there are indications that they may in some instances be more adapted and resistant to local diseases
(Currens et al., 1997). Thus, it is important to identify and conserve the native inland populations, and in some cases to replace hybridized populations with restored native populations. A number of programs in the region, in various subbasins, are currently conducting such efforts. Additional markers and markers with greater frequency differences between the inland and coastal types would improve the efficiency of such efforts.
The inland and coastal forms of rainbow trout have most often been differentiated using allozyme polymorphisms at lactate dehdyrogenase ( LDH-B2 ) and superoxide dismutase ( sSOD-1 ) loci detectable using protein electrophoresis, first demonstrated by Allendorf and Utter (1979). The patterns of differentiation have subsequently been found to be remarkably consistent across rainbow trout populations in the Columbia basin (Utter, 2001; summary data presented in Knudsen et al., 2002). As described by Knudsen et al. (2002), where the *100 allele is the most common type for the respective loci, inland rainbow trout (i.e., redband) populations have LDH-B2*76 (slow-migrating) allele frequencies that are usually greater than .250, and coastal rainbow trout populations have frequencies of less than .100.
Similarly, inland populations have sSOD-1*152 (rapidly-migrating) allele frequencies of less than .100, and coastal populations have frequencies of greater than .150. No other differences which are so consistent have been found differentiating the coastal and inland forms of rainbow trout. These LDH-B2 and sSOD-1 allele frequency differences have been successfully utilized to differentiate native and hybridized rainbow trout populations in widely dispersed portions of the Columbia River basin, including the Yakima (Campton and Johnstyon, 1985), Kootenai (Allendorf et al., 1980; Knudsen et al., 2002) and
Deschutes (Williams et al., 1997; Currens et al., 1997) subbasins. Mitochondrial DNA (McCusker et al.,
2000) and chromosome (Thorgaard, 1983) polymorphisms have been detected among rainbow trout populations, but they have thus far not bee found to be differentiated along inland versus coastal patterns.
Microsatellites show high levels of variation and can be locally useful in differentiating populations, but these markers thus far have not been demonstrated to show consistent inland versus coastal patterns

across the basin (e.g., Knudsen et al., 2002; Narum et al., 2004; Small et al., 2007). In addition, DNA typing in regional laboratories is increasingly moving toward SNP-based rather than microsatellite-based approaches.
The use of the lactate dehydrogenase and superoxide dismutase markers for identifying hybridization has several disadvantages, however. Most genetic sampling is now being conducted for
DNA rather than protein typing, and fish to be studied by allozyme assays must normally be sacrificed to obtain useful samples. We recently, with support from the Montana Department of Fish, Wildlife and
Parks, converted these two main allozyme assays ( LDH-B2 and sSOD-1 ) into Taqman (ABI) allelic discrimination SNP assays by identifying the single nucleotide polymorphism (SNP) differences in these genes responsible for the allozyme amino acid substitutions. The LDH-B2 test appears to be quite robust, while the sSOD-1 test needs further development because multiple DNA types are apparently associated with each allozyme type. Our conversions of allozyme tests to DNA tests are valuable developments because population profiling by PCR-based SNP assays is both more economical and efficient. However, additional DNA-based tests to distinguish inland and coastal types are needed.
There are numerous advantages to developing of PCR-based genotyping strategies. Samples for
DNA analysis are easier to collect and store because they can be collected as fin clips and stored in ethanol. This is a concern when small, sometimes threatened populations are being studied, and most of the genetic sampling in the basin is focused on DNA sampling. Our laboratory at Washington State
University has the expertise and experience to conduct these studies. We will genetically profile distinct inland and coastal populations of rainbow trout to identify DNA polymorphisms in genomic DNA and the expressed mRNA of genes using AFLP (amplified fragment length polymorphism) analysis of both genomic DNA and cDNA samples. Our laboratory has worked with AFLP for nine years and we have found it highly useful for sensitively distinguishing genetic differences within rainbow trout an dother salmonids. We would now like to apply the method to the broad issue of differentiating coastal and inland populations. This study will provide a method of converting population-typing genetic polymorphisms into simple, efficient PCR-based tests which can be used in a large number of laboratories to identify levels of hybridization within inland rainbow trout populations, and more broadly to study patterns of genetic differentiation within the rainbow trout species and potentially other salmonids.
C. Rationale and Significance to the Council’s Fish and Wildlife Program
The broad scientific principle being addressed by this project is the conservation of biological diversity (principle 6 in the Fish and Wildlife Program). As discussed above, introductions of non-native rainbow trout in the basin have in some cases disrupted the genetic makeup of the native redband populations. A high proportion of subbasins in the inland Columbia River basin have experienced some degree of hybridization between introduced, coastal hatchery rainbow trout and native inland redband rainbow trout. Many of these subbasins are above the current range of anadromous fishes, and thus these efforts to preserve and enhance the redband populations to some extent represent a substitution to mitigate the historic blockages, an established policy under the Fish and Wildlife Program.
D. Relationships to other projects
A number of other projects are currently sampling rainbow trout populations to determine the status of their genetic purity. Examples include studies in the Malheur, Spokane, Walla Walla, Hood and
Clearwater subbasins. A number of other studies are being considered (see list of related subbasin activities). Our laboratory is already in contact with key individuals involved in such analyses in Idaho
(Matt Campbell, Idaho Department of Fish and Game), Washington (Maureen Small, Washington
Department of Fish and Wildlife) and Montana (Jim Dunnigan, Montana Department of Fish, Wildlife and Parks) who have provided the samples described in the proposal. This established set of contacts should facilitate the rapid utilization of the tests that we develop.

E. Proposal objectives, work elements, methods and monitoring and evaluation
Objectives
The overall biological objective is to identify the level of hybridization between introduced and native rainbow trout in the interior Columbia basin. Unhybridized populations can be protected and hybridized populations may be replaced with populations having more local genetic content and better prospects for local adaptation. We will develop DNA tests for identifying the level of hybridization. Our specific experimental approaches (work elements) will be as follows:
1. Identify new DNA polymorphisms distinguishing the inland and coastal populations using the
AFLP (amplified fragment length polymorphism) technique. a. Identify genetic polymorphisms using genomic DNA samples as template. b. Identify genetic polymorphisms using the expressed gene transcript (cDNA) samples as template.
2. Convert population-typing genetic polymorphisms from an AFLP analysis into simple SNP allelic discrimination assays.
3. Validate the SNP allelic discrimination assays upon the individuals representing the inland vs. coastal populations.
Increasing the number of useful DNA markers differentiating inland and coastal rainbow trout would increase the accuracy of decisions about the genetic status of inland populations. We believe that methods which we have used successfully in our laboratory can help provide useful new markers for such efforts.
Methods
General methods common to both objectives (sample collection and processing):
Both liver (as a source of cDNA) and fin clips ( as a source of genomic DNA) will be collected from a limited subset of samples. Fin clips (for genomic DNA) will be collected from a broader set of samples. The genomic DNA samples will be analyzed using genomic AFLP and the cDNA samples will be analyzed using cDNA AFLP. Results from the cDNA-AFLP studies will then be applied to the broad set of samples.
At least twenty individuals each from six populations (three populations each from the coastal and inland types) will be sacrificed and liver samples will be collected for RNA recovery. The three coastal populations will include the Hayspur, Idaho hatchery population of the Idaho Department of Fish and
Game (already sampled), the Spokane hatchery of the Washington Department of Fish and Wildlife and one other coastal-type population (likely a coastal Washington steelhead). The three inland populations sampled will include the Dworshak, Idaho hatchery steelhead (already sampled) and two other populations (potentially the Wells Dam, Columbia River steelhead and Phalon Lake, Washington redband trout broodstock). Total RNA extracted from the liver will be used to synthesize cDNA. Fin clips will also be collected from these same individuals and used for preparing genomic DNA samples from all the individuals.
In addition, samples have already been obtained from the following populations as sources of genomic DNA. At least 20 samples have been obtained from all of the following populations, except an inland sample from Basin Creek, East Fork Yaak River, Montana, from which only fifteen samples were obtained. Samples have been obtained from six additional inland populations, including Little Sheep
Creek (Imnaha River), Oregon; Rapid River (Salmon River), Idaho; South Fork Salmon River, Idaho;

Pahsimeroi hatchery (Snake River system), Idaho; N.F. Little Deep Creek (Spokane River), Washington; and W.F. Trout Creek (Kettle River), Washington. Samples have also been obtained from South Tacoma hatchery (California origin), Washington; Upper Dosewallips River (Olympic Peninsula), Washington; and Kalama River, Washington. We thank the Idaho Department of Fish and Game, the Nez Perce Tribe and the Washington Department of Fish and Wildlife for generously providing the samples.
Identification of new DNA polymorphisms using the AFLP technique
Distinctive, individually characteristic genetic profiles are revealed using Amplified Fragment
Length Polymorphisms (AFLP) analysis. This technique utilizes PCR (polymerase chain reaction) to rapidly detect DNA sequence differences among DNA samples. Conducting an AFLP analysis involves first digesting a DNA sample with two different restriction enzymes, followed by ligating restriction sitespecific adaptors to the cut sites. This prepared genomic “restriction/ligation” template is then selectively
PCR-amplified using primers specific to the distinct adaptor sequences which also overlap the first three internal bases (“+3”) of the genomic fragment to be amplified. This process of “selective amplification” yields about 100 amplified fragments for any specific “+3” AFLP amplification, which are then visualized by acrylamide gel analysis. However each prepared genomic template allows for 64 (=4X4X4) possible primer options specific to either distinct adaptor, allowing for tremendous screening potential through this analytical technique.
The complex, bar-code-like pattern of bands visualized by gel will typically show 10-20 band differences between any two genomic DNA samples from the same species. AFLP has been used most widely in plant genetics laboratories. Our laboratory has used this method extensively for DNA typing.
This has included applications to genetic mapping (e.g., Young et al., 1998; Nichols et al., 2003) and to the isolation of sex-associated DNA markers. An example of the application of AFLP in the recovery of a specific genomic fragment is seen in the AFLP gel-image comparing sex, in Figure 1 derived from
Brunelli and Thorgaard, 2004. The arrow in the figure is pointing at a genetic difference which distinguishes male from female Chinook salmon by AFLP. This analysis yielded sequence information allowing easy and effective PCR-based discrimination of male derived DNA from female derived DNA
(e.g., Brunelli and Thorgaard, 2004) Similar results have been obtained with rainbow trout (Felip et al.,
2004, 2005). We anticipate that this method can also be used to develop useful DNA markers differentiating the inland and coastal rainbow trout. The method can be applied to both genomic (total nuclear) DNA and to cDNA (DNA sequences corresponding to genes expressed in a specific tissue). a. Genomic DNA AFLP
In order to maximize the detection of lineage-specific genetic differences while minimizing individual allelic variance, AFLP analysis will be conducted on pooled genomic DNA samples from each population for identification of polymorphisms associated with the inland versus coastal rainbow trout.
From each population of twenty to forty individuals, we would prepare four separate normalized pools.
The use of pools allows efficient screening while also providing replication to correct for any problems with experimental repeatability. Our total screen for a given set of AFLP primers would thus involve 48 lanes (four pools per population times twelve (six inland, six coastal) populations) and thus could be analyzed on a single gel with 48 lanes.
After conducting the AFLP typing, informative bands are sequence-characterized, and SNP allelic discrimination assays are developed to characterize the genomic sequence differences seen in the
AFLP analysis. This process involves characterizing flanking genomic sequence to the polymorphic
AFLP restriction-site, which we have accomplished by both inverse-PCR methods and PCR-based library

Figure 1. Genomic AFLP patterns in Chinook salmon revealing sex-associated DNA fragments.

screens. We have gone through this process successfully for developing single-locus sex markers for chinook salmon (Brunelli and Thorgaard, 2004) and rainbow trout (Felip et al., 2004).
Although it was not a focus of that study, a previous AFLP tying study to identify rainbow X cutthroat hybrids (Young et al., 2001) did include an inland rainbow trout population (Dworshak steelhead) and found clear indications that useful markers differentiating coastal and inland rainbow trout should be readily detectable. This supports the feasibility of our approach.
Single nucleotide differences detected using the pooled samples will then be analyzed in individuals and allele frequencies described in our full set of samples. b. cDNA AFLP
Population-specific genetic polymorphisms can be detected by AFLP comparison of cDNA templates as well as genomic DNA templates. The cDNA-AFLP technique compares differences in expressed gene sequences by the same methodology described above for genomic DNA samples. The significant difference is that rather than using genomic DNA as a template, the cDNA-AFLP approach uses expressed gene sequences by reverse-transcribing mRNAs into cDNA copies, which are then utilized in the AFLP analysis. The cDNA templates will be pooled to generate 4 homogeneous cDNA pools per population, as described above for genomic DNA. With three inland and three coastal populations sampled for liver, a total of 24 cDNA-AFLP template pools will be developed. This will allow two reaction sets to be analyzed per 48-well gel.
Differences detected using this approach will reflect allelic frequency differences in expressed genes between these populations. The potential for such differences is already evident from the allozyme analyses. The power of the cDNA-AFLP analysis is based on the fact that the detection of allelic fixation will no longer depend upon differences in protein mobility in allozyme gels, which only detect amino acid charge difference substitutions in the small subset of genes for which histochemical staining reactions have been developed. Any genetically-fixed single nucleotide polymorphism (SNP) which affects an
AFLP-selected restriction site within the coding region of any gene expressed in the tissue of choice (in our study, liver) could potentially be detected. The population-distinguishing fragment can then be gel purified, sequenced, and characterized for coding content by comparison to existing DNA sequence databases (e.g., TIGR and Genbank). Sequence-characterized polymorphic fragments should provide enough information for NCBI-BLAST analysis and gene identification. This analysis directly reveals flanking sequence necessary for SNP assay development and therefore circumvents the cloning steps necessary in converting genomic DNA AFLP polymorphisms into SNP assays.
Any population-characterizing polymorphic cDNA-AFLP fragment will then be used to characterize individuals from the inland vs. coastal populations described. This will entail designing primers which will amplify across the region containing the AFLP polymorphism, facilitated by using the gene sequence information available through the DNA sequence databases. The fact that virtually all the polymorphisms detected using the cDNA-AFLP screening process will be in single copy, expressed genes is advantageous because the allelic difference will not be derived from repetitive sequences, thus simplifying marker development and interpretation.
Less than a hundred genes have been screened using allozyme methods, and two showed useful differentiation of the inland and coastal rainbow trout. With the cDNA-AFLP method we can potentially screen all of the extremely large numbers of genes expressed in any given tissue and will no longer be restricted to the genes detectable using histochemical staining methods. It is noteworthy that most SNPs found in human alleles involve mutation of CpG dinucletide sites. The AFLP processes we will employ incorporate this preferential mutability by focusing on restriction enzymes (TaqI and MspI) targeting sites containing CpG. If a similar proportion of the genes showing useful differences with allozymes (>2%) that we screen with cDNA-AFLP show useful differences between the groups, we are likely to detect a large number of new DNA markers which will facilitate differentiating the inland and coastal groups and identifying hybridized populations.
After markers have been identified by cDNA-AFLP, we will test them on genomic samples from the wider set of samples which we have available. This, along with similar experiments using markers

developed from the genomic AFLP studies, represent the monitoring and evaluation phase of the project.
Allele frequencies will be described for our full set of samples. The markers would then be available for use by the management agencies we are already in communication with.
Ideally, we hope to identify a significant number of new markers that are diagnostic or have high frequency differences between the inland and coastal populations. The more such markers that can be developed, the greater the power that DNA analyses can bring to the assessment of relative levels of hybridization and purity of the populations.
Overall project flow and timeline
We are requesting 18 months of support for the project. The first twelve months will be the experimental phase and the final six months will focus on analysis and completion of reports and manuscripts for publication. We anticipate that our experimental effort would be distributed roughly equally between genomic AFLP and cDNA-AFLP. We will also simultaneously convert characterized AFLP differences into SNP assays which will be immediately evaluated for population discriminating utility upon the available samples.
F. Facilities and Equipment
All of the essential equipment for the project is already in place. This includes six Thermolyne thermocyclers for PCR, four Hoefer agarose gel electrophoresis units, four large polyacrylamide sequencing gel units for use in AFLP or microsatellite analyses, power supplies for the electrophoresis units, a temperature controlled bath with circulator for cooling the agarose gel units during long runs, two
Robbins hybridization incubation chambers for Southern blot analysis, two microcentrifuges, two clinical centrifuges, balances, pH meter, VWR drying oven, UV irradiation box for cross-linking DNA to membranes, transilluminator, Gel Logic 200 digital imaging system, two water baths, and miscellaneous glassware and chemicals. We also access to core sequencing and fragment analysis facilities on campus.
A Storm 860 phosphoimager/ fluorescence scanner and a Storm Typhoon scanner are located in our building and are key pieces of equipment that we use for imaging AFLP gels. An Applied Biosystems
7300 Real-Time PCR System is also available for use in SNP typing and gene expression studies.

G. Literature Cited
Allendorf, F.W., and F.M. Utter, 1979. Population genetics. Pages 407-454. in W.S. Hoar, D. J. Randall and J.R. Brett, Editors. Fish Physiology, volume 8. Academic Press, New York.
Allendorf, F.W., D.M. Espeland, D.T. Scow and S. Phelps, 1980. Coexistence of native and introduced rainbow trout in the Kootenia River drainage. Proc. Mont. Acad. Sci. 39: 28-36.
Behnke, R.J., 1992. Native trout of western North America. American Fisheries Society, Monograph 6.
Bethesda, Maryland.
Brunelli, J.P. and G.H. Thorgaard, 2004. A new Y-chromosome-specific marker for Pacific salmon.
Trans. Amer. Fish Soc. 133: 1247-1253.
Brunelli, J.P., Robison, B.D., and G.H. Thorgaard, 2001. Ancient and recent duplications of the rainbow trout Wilms' tumor gene. Genome 44: 1-8.
Campton, J.E. and J.M. Johnston, 1985. Electrophoretic evidence for a genetic admixture of native and nonnative rainbow trout in the Yakima river, Washington. Trans. Amer. Fish. Soc. 114: 782-793.
Currens, K.P., A.R. Hemmingsen, R.A. French, D.V. Buchanan, C.B. Schreck and H.W. Li, 1997.
Introgression and susceptibility to disease in a wild population of rainbow trout. N. Am. J. Fish. Manage.
17: 1065-1078.
Dillon, J.C., D.J. Schill and D.M. Teuscher, 2000. Relative return to creel of triploid and diploid rainbow trout stocked in eighteen Idaho streams. N. Am. J. Fish. Manage.20: 1-9..
Felip, A., A. Fujiwara, W.P. Young, P.A. Wheeler, M. Noakes, R.B. Phillips and G.H. Thorgaard, 2004.
Polymorphism and differentiation of rainbow trout Y chromosomes. Genome 47: 1105-1113.
Felip, A., W.P. Young, P.A. Wheeler and G.H. Thorgaard, 2005. An AFLP-based approach for the identification of sex-linked markers in rainbow trout ( Oncorhynchus mykiss ). Aquaculture 247: 35-43.
Knudsen, K.L., C.C. Muhlfeld, G.K. Sage and R.F. Leary, 2002. Genetic sstructure of Columbia River redband trout population sin the Kootenai River drainage, Montana, revealed by microsatellite and allozyme loci. Trans. Amer. Fish. Soc. 131: 1093-1105.
McCusker, M.R., E. Parkinson and E. B. Taylor, 2000. Mitochondrial variation in rainbow trout
Oncorhynchus mykiss across its native range: testing biogeographical hypotheses and their relevance to conservation. Mol. Ecol. 9: 2089-2108.
Narum, S.R., C. Contor, A. Talbot and M. S. Powell, 2004. Genetic divergence of sympatric resident and anadromous forms of Oncorhynchus mykiss in the Walla Walla River, U.S.A.. J. Fish Biol. 65: 471-488.
Nichols, K.M., W.P. Young, R.G. Danzmann, B.D. Robison, C. Rexroad, M. Noakes, R.B. Phillips, P.
Bentzen, I. Spies, K. Knudsen, F.W. Allendorf, B.M. Cunningham, J. Brunelli, H. Zhang, S. Ristow, R.
Drew, K.H. Brown, P.A. Wheeler and G.H. Thorgaard, 2003. A consolidated linkage map for rainbow trout ( Oncorhynchus mykiss ). Anim. Genet. 34: 102-115.

Small, M.P., J.G. McClellan, J. Loxterman, J. Von Bargen, A. Frye and C. Bowman, 2007. Fine-scale population structure of rainbow trout in the Spokane river drainage in relation to hatchery stocking and barriers. Trans. Amer. Fish. Soc. 136: 301-317.
Thorgaard, G. H., 1983. Chromosomal differences among rainbow trout populations. Copeia 1983: 650-
662.
Utter, F., 2001. Patterns of subspecific anthropogenic introgression in two salmonid genera. Rev. Fish
Biol. Fish. 10: 265-279.
Williams, R.N., R.F. Leary and K.P. Currens, 1997. Localized genetic effects of a long-term hatchery stocking program on resident rainbow trout in the Metolius River, Oregon. N. Am. J. Fish. Manage. 17:
1079-1093..
Young, W.P., P.A. Wheeler, V.H. Coryell, P. Keim and G. H. Thorgaard, 1998. A detailed linkage map of rainbow trout produced using doubled haploids. Genetics 148: 839-850.
Young W.P., C.O. Ostberg CO, P. Keim and G.H. Thorgaard, 2001. Genetic characterization of hybridization and introgression between anadromous rainbow trout ( Oncorhynchus mykiss irideus ) and coastal cutthroat trout ( O. clarki clarki ). Mol. Ecol. 10:921-30.
H. Key Personnel
Gary Thorgaard, Professor, 5% effort to be provided at no charge during the academic year during the project period. Principal investigator. Will provide overall oversight for the project. Will be responsible for securing the samples, insuring that the project is carried out and that the results are disseminated in the fisheries and scientific communities.
Joseph Brunelli, Assistant Research Professor. 1.0 FTE to be funded for 18 months. Co-principal investigator. Will provide his molecular biology expertise to insure that the specific experimental objectives are carried out successfully.

Gary H. Thorgaard, Ph.D.
EDUCATION
Ph. D. 1977 University of Washington - Genetics
B.S. 1973 Oregon State University – Zoology (Honors)
PROFESSIONAL EXPERIENCE
Washington State University, Pullman, Washington
August 1999- August 2001, August 2005- present, Director, School of Biological Sciences.
August 1991-August 1999, Chair, Department of Zoology
August 1999- present, Professor, School of Biological Sciences
August 1990-August 1999, Professor of Genetics and Zoology
August 1986-August 1990, Associate Professor of Genetics and Zoology
September 1979- August 1986, Visiting Assistant Professor and Assistant Professor
PREVIOUS EMPLOYMENT
Postdoctoral Fellow, 1978-79, University of California, Davis
Graduate assistant, 1973-1977, University of Washington
PROFESSIONAL EXPERTISE
Professional expertise includes many areas in trout and salmon genetics, including induced polyploidy, gynogenesis and androgenesis, DNA polymorphisms, genetic conservation, gene banking, genetic analysis and mapping.
SELECTED PUBLICATIONS (from a total of over 130)
Brown, K.H., S.J. Patton, K.E. Martin, K.M. Nichols, R. Armstrong, and G.H. Thorgaard, 2004. Genetic analysis of interior Pacific Northwest Oncorhynchus mykiss reveals apparent ancient hybridization with westslope cutthroat trout. Trans. Amer. Fish. Soc. 133: 1078-1088.
Nichols, K.M., W.P. Young, R.G. Danzmann, B.D. Robison, C. Rexroad, M. Noakes, R.B. Phillips, P.
Bentzen, I. Spies, K. Knudsen, F.W. Allendorf, B.M. Cunningham, J. Brunelli, H. Zhang, S.
Ristow, R. Drew, K.H. Brown, P.A. Wheeler and G.H. Thorgaard, 2003. A consolidated linkage map for rainbow trout ( Oncorhynchus mykiss ). Anim. Genet. 34: 102-115.
Thorgaard, G.H., G.S. Bailey, D. Williams, D.R. Buhler, S.L. Kaattari, S.S. Ristow, J.D. Hansen, J.R. Winton, J.L.
Bartholomew, J.J. Nagler, P.J. Walsh, M.M. Vijayan, R.H. Devlin, R.W. Hardy, K.E. Overturf, W.P.
Young, B.D. Robison, C. Rexroad III, Y. Palti, 2002. Status and opportunities for genomics research with rainbow trout. Comp. Biochem. Physiol. B., 133: 609-646.
Thorgaard, G.H., P.A.Wheeler, J.G. Cloud and T.R. Tiersch, 1998. Gene banking efforts for endangered fishes in the United States. In: Action before Extinction: An International Conference on
Conservation of Fish Genetic Diversity. B. Harvey, C. Ross, D. Greer and J. Carolsfeld, eds.
World Fisheries Trust, Victoria, British Columbia, pp. 181-185,
Cloud, J.G. and G.H. Thorgaard, eds., 1993. Genetic Conservation of Salmonid Fishes. NATO ASI
Series A: Life Sciences Vol. 248. Plenum Press, New York, 314 pp.

Joseph Peter Brunelli, Ph.D.
EDUCATION
Ph.D. 1994 Washington State University, Genetics & Cell Biology
B.S. 1989 University of Wyoming, Biochemistry
PROFESSIONAL EXPERIENCE:
2003- Assistant Research Professor, School of Biological Sciences, Washington State University
2002- Visiting Assistant Professor, School of Biological Sciences, Washington State University
1996- Postdoctoral Research Associate, School of Biological Sciences, Washington State University
1994- Postdoctoral Research Associate, Genetics and Cell Biology, Washington State University,
PROFESSIONAL EXPERTISE
Professional expertise spans a wide variety of molecular techniques for analysis of DNA and gene expression. This experience covers most fundamental methodologies used for DNA and RNA analyses, including genotyping, cloning strategies and DNA sequencing, genomic and cDNA library constructions,
Amplified Fragment Length Polymorphism analysis (AFLP), Methylation-Sensitive AFLP, cDNA-AFLP,
RT-PCR gene expression analysis.
SELECTED PUBLICATIONS
1. Brunelli, J.P. and G.H. Thorgaard, 2004. A new Y-chromosome-specific marker for Pacific salmon.
Transactions of the American Fisheries Society. Vol 133(5): 1247–1253.
2. Brunelli, J.P., Robison, B.D., and G.H. Thorgaard, 2001. Ancient and recent duplications of the rainbow trout Wilms' tumor gene. Genome 44: 1-8.
3. Brunelli JP, Thorgaard GH. 1999. Sequence, expression and genetic mapping of a rainbow trout retinoblastoma cDNA. Gene Jan 21;226(2):175-80.
4. Brunelli JP, Pall ML. 1994. Lambda/plasmid vector construction by in vivo cre/lox-mediated recombination. Biotechniques. Jun;16(6):1060-4.
5. Brunelli JP, Pall ML. 1993. A series of yeast/Escherichia coli lambda expression vectors designed for directional cloning of cDNAs and cre/lox-mediated plasmid excision. Yeast. Dec;9(12):1309-18.